The long term objective of this project is to develop a method that will lead to high throughput production of conditional knock out mice. The immediate goal of this 2 year project is to develop and optimize a novel procedure that may save 6 - 8 months in each conditional knock out project while cutting cost to about half. This technology has two main features: (1) the development of a latent targeting cassette, which can be applied to any gene and activated by Cre to disrupt the target gene. This use of this cassette will eliminate the tedious process of individually inserting loxP sites; (2) the formulation of a novel cassette that allows highly efficient removal of the neomycin resistance marker without ES cell transfection/single colony selection or breeding with Flpe transgenic mice. The project has two specific aims: (1) Test and optimize different components that will contribute to the disruption of a target gene, and construct a conditional gene disruption cassette that is generally applicable to all genes of known structural organizations; (2) Test the utility of the technology by constructing a Csk (C- terminal Src kinase) conditional knock out allele, and analyze vascular defects due to conditional Csk disruption in early embryos and adult tissues. Csk is chosen to test the technology because it has been found previously that Csk-/- (null) embryos display severe vascular defects. By comparing mutant phenotypes in Csk-/- and Csk conditional knock out embryos, it can be concluded if the proposed gene targeting cassette can indeed effectively disrupt gene function. Furthermore, the Csk conditional knock out mice will also be used to investigate the role of Csk in the adult vascular system and to determine whether endothelial Csk is directly required for angiogenesis. This research will enhance the pace of biomedical research in many fields by accelerating the speed of generating conditional knock out alleles, and directly contribute to angiogenesis research by Csk conditional knock out. Thus, the proposed work is relevant to the mission of NIH, and in particular the NHLBI. Studies of human diseases (such as cardiovascular diseases) are heavily reliant on inducible mutant mouse models generated by genetic manipulation. However, current technologies for the introduction of inducible mutations into mice are very time consuming and expensive. This project aims at developing a novel technology that will significantly simplify this procedure and therefore accelerate biomedical research. [unreadable] [unreadable] [unreadable]